The present invention, in some embodiments thereof, relates to novel biomaterials and, more particularly, but not exclusively, to malleable hybrid hydrogels made of peptide(s) and polymer(s).
Biomaterials for tissue engineering are required to possess unique characteristic features for allowing proper biocompatibility, supporting growth and handy biomechanical properties enabling injectability and malleability of the matrix substances.
Development of malleable polymeric nanofiber constructs is of a great scientific and technological interest due to their wide-range applications in biomedicine and biotechnology. Particularly, composite nanofibers derived from natural and synthetic polymers, combining the favorable biological properties of the natural polymer and the mechanical strength of the synthetic polymer, represents a major advantageous advancement in tissue engineering and regenerative medicine.
Hydrogels are determined as polymer networks that are insoluble in water, where they swell to an equilibrium volume but retain their shapes. Hydrogels are of great interest as a class of materials for tissue engineering and regenerative medicine, as they offer 3D scaffolds to support the growth of cultured cells. In terms of material requirements, hydrogels have long received attention because of their innate structural and compositional similarities to the extracellular matrix and their extensive framework for cellular proliferation and survival.
A variety of natural polymers, including agarose, collagen, fibrin, alginate, gelatin, chitosan and hyaluronic acid (HA), may be used as hydrogel-forming materials [Almany and Seliktar 2005, Biomaterials 26: 2467-2477]. These polymers are appealing for medical use owing to their similarity to the natural extracellular matrix (ECM), which allows cell adhesion, migration and proliferation, while maintaining very good biocompatible and biodegradable qualities.
Another class of building blocks for hydrogel formation includes synthetic materials, such as poly(ethylene oxide), poly(vinyl alcohol) and poly[furmarate-co-(ethylene glycol)]. These synthetic building blocks offer controllability and reproducibility, but several drawbacks include their production methods that sometimes involve the use of extreme temperatures and pressures and the use of complex techniques, as well as low biocompatibility of the products.
Hydrogels can be prepared either by production of chemical gels or by production of physical gels. Chemical hydrogels are produced by crosslinking starting materials, through chemical or polymerization reactions, and hence involve covalent linking of the hydrogel networks. Physical hydrogels are receiving a great attention since their production does not involve chemical reactions, a fact which is advantageous in the context of encapsulation of cells and other sensitive molecules therein, since laborious removal of toxic or extremely reactive molecules used to initiate chemical crosslinking reactions is circumvented. In physical hydrogels, the networks are held together by molecular entanglements, and/or secondary forces including electrostatic forces, hydrogen-bonding forces or hydrophobic forces [Campoccia et al. 1998 Biomaterials 19: 2101-2127; Prestwich et al. 1998 J. Controlled Release 53: 93-103].
Hyaluronic acid (HA) is a high molecular weight unsulfated glycosaminoglycan (GAG) present in all mammals. HA is composed of repeating disaccharide units composed of (β-1,4)-linked D-glucuronic acid and (β-1,3)-linked N-acetyl-D-glucosamine (see, FIG. 1A). GAG, a major component of the native extracellular matrix (ECM), is known to support enhanced cell attachment and proliferation and to improve the material's cellular and tissue biocompatibilities. HA in the body occurs as its sodium salt form hyaluronate and is found in high concentrations in the fetus, umbilical cord, and in several soft connective tissues of adults, including skin, synovial fluid, and vitreous humor.
Being a highly hydrated, negatively charged, linear biodegradable and biocompatible natural polymer, characterized by high viscoelastic and space filling properties, HA is highly useful for tissue engineering applications. The advantageous rheological features of HA are exploited, for example, in the application of hyaluronan for ophthalmic surgery [Pape and Balazs 1980, Ophthalmology 87(7): 699-705.], in the cosmetic field [Duranti et al 1998 Dermatol. Slug. 24: 1317-1325], and in the intra-articular treatment of osteoarthritis [Goa and Benfield 1994 Drugs 47(3): 536-566].
However, the use of HA is limited by its poor mechanical strength and by its rapid in vivo enzymatic digestion by hyaluronidase. Overcoming these limitations can be made by introducing synthetic cross-linkers, for providing strengthen HA composite with reduced biodegradation rate [Leach et al. 2003 Biotechnol. Bioeng. 82(5), 578-589; Lu et al. 2008 J. Biomater. Sci. Polym. 19:1-18; and Pitarresi et al. 2008 J. Biomed. Mater. Res, Part A 84A(2): 413-424], and/or by employing specific or non-specific inhibitors of hyaluronidase.
Self-assembled nanotubes and hydrogels made of short (aromatic) peptides have been disclosed in Mahler et al. Adv. Mater. 18: 1365-1370, and in WO 2007/0403048, WO 2004/052773 and WO 2004/060791. An exemplary building block for forming such nanotubes and hydrogels is Fmoc-FF.
Fmoc-FF is a protected dipeptide, which was shown to self-assemble into discrete, well-ordered nanotubes and to form hydrogels in the macrostructure. The diphenylalanine peptide (FF) is the natural core recognition motif of the amyloid-βpolypeptide. The Fmoc group (9-fluorenylmethoxycarbonyl) is widely used as a synthetic protecting group in peptide synthesis and it was reported by Burch et al. that a number of Fmoc-amino acids show anti-inflammatory properties [Burch et al. 1991 Proc. Natl. Acad. Sci. U.S.A. 88: 355-359].
The efficient self-assembly, under mild conditions, of Fmoc-FF into a hydrogel which exhibits remarkable physical properties has been reported (see, Mahler et al., supra). In spite of the short building-block size, the obtained hydrogel was characterized by physical properties that exceed those of hydrogels formed from longer polypeptides.
Hybrid composite hydrogels are reviewed, for example, in Jia and Kiick in Macromol Biosci. 2009 Feb. 11; 9(2): 140-156, and references cited therein. Several hybrid hydrogels made of hyaluronic acid and polymers such as PEG, chitosan, cellulose and alginate have been reported. Hybrid hydrogels made of hyaluronic acid and other polysaccharides and proteins such as collagen, gelatin and fibrin have also been reported. Hydrogel matrices made of hyaluronic acid derivatized by a cell adhesive peptide fragment are disclosed, for example, in U.S. Pat. Nos. 5,834,029 and 6,156,572. Hydrogels made of hyaluronic acid modified with Nodo-66 antagonist have been reported in Hou et al., J. Neurosci. Met. 137:519-529, 2005.
Additional background art includes Yang et al., Biomedical Materials, 2001, 6, 025009; Kim et al., Acta Biomater. 2008, 4(6):1611-1619; and Park et al., Key engineering materials, Vols. 342-343 (2007), pp. 153-156.